Fluorescence microscopy is often used in the fields of molecular biology, biochemistry and other life sciences. One such use is in identifying a specific antigen using antibodies. Antibodies are proteins produced by vertebrates as a defense against infection. They are made of millions of different forms, each having a different binding site and specifically recognizing the antigen that induces its production. To identify an antigen, a sample of cells is provided that contains specific antibodies coupled to a fluorescent dye. The cells are then assessed for their fluorescence. Taking advantage of the precise antigen specificity of antibodies, the cells having fluorescent properties are known to contain a specific antigen.
Originally, the fluorescence of cells was assessed manually by visual inspection, using conventional microscopy. This proved time-consuming and costly. The need for high-speed automated systems became manifest. Many high-speed imaging systems, such as confocal microscopes, are available for assaying cell samples. The illumination and collection optics, along with their relative geometry, determine in large part the parameters of the other system elements.
A prior art high-speed imaging system is shown in FIG. 1 and includes an F-.THETA. objective 10 positioned above a sample 11 so that the surfaces of the objective are perpendicular to the sample's normal. A laser light source 12 produces a beam 13. The objective 10 directs the beam 13 to illuminate a spot on the sample's surface. An oscillating reflective surface 14 is disposed at the pupil 15 of the system, between the light source 12 and the objective 10, to deflect the beam 13 back and forth along one axis. The sample is placed on a table to move the sample in a direction perpendicular to the first scan direction, thereby resulting in a two dimensional scan pattern on the sample's surface. The objective is not designed for coaxial collection resulting in light reflected from the sample surface being collected by a condenser assembly 16 that is separate and apart from the objective. Such a geometry results in increased system footprint, increased optical complexity and a limitation of solid angle collection. The collected light is then imaged on a photo-detector 17. The design of a classical F-.THETA. lens is primarily for monochromatic illumination. As a result, such lenses lack good polychromatic performance. Therefore, the objective 10 manifests lateral and axial chromatic aberrations over a broad band of wavelengths.
A prior art high-speed imaging system, similar to that described with respect to FIG. 1, is disclosed by Richard L. Shoemaker et al., in "An Ultrafast Laser Scanner Microscope for Digital Imaging Analysis", IEEE Transactions on Biomedical Engineering, Vol. BME-29, No. 2, February 1982, pp. 82-91. The principal difference between these two systems concerns the scanning device. Instead of a galvanometric scanner, Shoemaker et al. require the use of a rotating polygon mirror to scan the spot over the sample's surface.
Another prior art high-speed imaging system is disclosed in U.S. Pat. No. 4,284,897 by Sawamura et al., in which laser light is reflected through two galvanometric mirrors and one dichroic mirror to direct a beam through an objective and illuminate a spot on a sample's surface. The galvanometric mirrors are swung in appropriate directions to allow the spot to scan over the entire surface of the sample. In response to the illuminating spot, the sample emits fluorescence light. The objective, serving as a condenser lens, transmits the light back through a first dichroic mirror. Positioned behind the first dichroic mirror is a second dichroic mirror that splits the fluorescent light into a light produced by a first probe at a first wavelength and light produced by a second probe at a second wavelength. The first and second wavelengths are transmitted to respective photo-detectors.
U.S. Pat. No. 5,296,700 to Kumagai discloses a fluorescent confocal microscope which includes, in pertinent part, an intermediary optical system disposed between a pair of scan mirrors and an objective optical system. The intermediary optical system is designed to cancel chromatic aberrations of magnification introduced by the objective optical system.
U.S. Pat. No. 5,260,578 to Bliton et al. discloses a scanning confocal microscope which includes, in pertinent part, two beam sources. One beam source produces ultra violet light. One beam source produces visible light. An optical assembly is included in the common optical train to correct chromatically induced scanning errors.
U.S. Pat. No. 5,381,224 by Dixon et al. discloses a scanning laser imaging system which allows simultaneous confocal and non-confocal imaging of reflected light. The system includes, in pertinent part, a laser producing a beam which traverses a beam expander and impinges upon a single mirror disposed in an optical axis, which is defined by an objective lens. The objective lens directs the beam onto a sample, which is disposed upon a moveable stage. The mirror scans the beam along a first direction, and the stage moves the sample along a second direction, transverse to the first direction. In this manner, the beam scans across the sample in two directions. Disposed between the objective and the sample is a beam splitter designed to collect light emitted from the sample. The beam splitter directs a portion of light emitted from the sample onto a condenser lens, which in turn directs it onto a non-confocal detector. A portion of the light collected by the beam splitter is directed along the same path as the beam, but in an opposite direction, forming a retro-beam. The retro-beam impinges upon a second beam splitter, positioned between the scan mirror and the laser. The second beam splitter directs the light onto a focusing lens. The focusing lens is positioned proximate to a field stop, having an aperture. The aperture is confocal to the light emitted from the sample and selectively restricts light in the retro-beam from reaching the detector. Light traversing the aperture impinges upon a confocal detector.
A disadvantage of the prior art systems is that additional optics are required to correct optical aberrations over a scan field and to efficiently collect light emitted from a sample, thereby increasing the systems' cost and size.
What is needed, therefore, is to provide a high-speed, low cost, laser scanning system that will provide point by point image of a sample on both a micro and macro scale.
A further need exists to provide an imaging system of a substantially smaller size than the prior art systems that affords a larger scan field than existing coaxial illumination and collection systems.